PCR not working - overhangs too long? (Mar/11/2010 )

Hi guys I want to subclone a gene another vector. I had to add long overhangs because I want to add a tag. I have put 25 bp annealing with the insert and 45bp overhang. I now have one primer which is 40 bp and another that is 70 bp (bearing the tag). I thought the PCR would work anyway cause the region that anneals is for both 25 bp. But it did not work. Obviously I have calculated the Tm considering only the region that anneals Any suggestions is very much appreciated!

Can we have a look see at the sequence? Perhaps the Tm was miss calculated?
Could you provide us will all the detail of exactly what you did, every detail. The PCR amplification conditions, polymerase type used, length of desired PCR product.

Often when a PCR fails, the cause is the template. The template may have been degraded, or more often is simply dirty.

Run some of your template DNA onto a gel to make sure there is still intact DNA. You should see a single high molecular weight band. if you see a smear your DNA is degraded

For a sample which is potentially dirty used phenol-chloroform to clean up your DNA. If that was already done, you could try diluting an aliquot of your DNA template 20 to 50 times with distilled water. Any contaminant in the template will be diluted out, and the PCR 's nature will amplify the DNA.

PS: Also make sure you have the right template and that the primer sequence is correct. it can one of those blind moments.

i used primers with 90bp overhang, so far it's still ok.

maybe there is problem with ur primers?

Thanks guys, problem solved. I have played around a lot with the annealing T and it worked.

badguy on Mar 16 2010, 08:23 PM said: